RESUMO
The conjunctival epithelium covering the eye contains two main cell types: mucus-producing goblet cells and water-secreting keratinocytes, which present mucins on their apical surface. Here, we describe long-term expanding organoids and air-liquid interface representing mouse and human conjunctiva. A single-cell RNA expression atlas of primary and cultured human conjunctiva reveals that keratinocytes express multiple antimicrobial peptides and identifies conjunctival tuft cells. IL-4/-13 exposure increases goblet and tuft cell differentiation and drastically modifies the conjunctiva secretome. Human NGFR+ basal cells are identified as bipotent conjunctiva stem cells. Conjunctival cultures can be infected by herpes simplex virus 1 (HSV1), human adenovirus 8 (hAdV8), and SARS-CoV-2. HSV1 infection was reversed by acyclovir addition, whereas hAdV8 infection, which lacks an approved drug therapy, was inhibited by cidofovir. We document transcriptional programs induced by HSV1 and hAdV8. Finally, conjunctival organoids can be transplanted. Together, human conjunctiva organoid cultures enable the study of conjunctival (patho)-physiology.
Assuntos
Túnica Conjuntiva , Células Caliciformes , Humanos , Camundongos , Animais , Túnica Conjuntiva/metabolismo , Células Caliciformes/metabolismo , Epitélio , Interleucina-13 , Homeostase , OrganoidesRESUMO
ABSTRACT: Regulation of lineage biases in hematopoietic stem and progenitor cells (HSPCs) is pivotal for balanced hematopoietic output. However, little is known about the mechanism behind lineage choice in HSPCs. Here, we show that messenger RNA (mRNA) decay factors regnase-1 (Reg1; Zc3h12a) and regnase-3 (Reg3; Zc3h12c) are essential for determining lymphoid fate and restricting myeloid differentiation in HSPCs. Loss of Reg1 and Reg3 resulted in severe impairment of lymphopoiesis and a mild increase in myelopoiesis in the bone marrow. Single-cell RNA sequencing analysis revealed that Reg1 and Reg3 regulate lineage directions in HSPCs via the control of a set of myeloid-related genes. Reg1- and Reg3-mediated control of mRNA encoding Nfkbiz, a transcriptional and epigenetic regulator, was essential for balancing lymphoid/myeloid lineage output in HSPCs in vivo. Furthermore, single-cell assay for transposase-accessible chromatin sequencing analysis revealed that Reg1 and Reg3 control the epigenetic landscape on myeloid-related gene loci in early stage HSPCs via Nfkbiz. Consistently, an antisense oligonucleotide designed to inhibit Reg1- and Reg3-mediated Nfkbiz mRNA degradation primed hematopoietic stem cells toward myeloid lineages by enhancing Nfkbiz expression. Collectively, the collaboration between posttranscriptional control and chromatin remodeling by the Reg1/Reg3-Nfkbiz axis governs HSPC lineage biases, ultimately dictating the fate of lymphoid vs myeloid differentiation.
Assuntos
Medula Óssea , Células-Tronco Hematopoéticas , Linhagem da Célula/genética , Células-Tronco Hematopoéticas/metabolismo , Medula Óssea/metabolismo , Hematopoese/genética , RNA Mensageiro/metabolismo , Diferenciação Celular/genéticaRESUMO
Cancer mortality primarily stems from metastatic recurrence, emphasizing the urgent need for developing effective metastasis-targeted immunotherapies. To better understand the cellular and molecular events shaping metastatic niches, we used a spontaneous breast cancer lung metastasis model to create a single-cell atlas spanning different metastatic stages and regions. We found that premetastatic lungs are infiltrated by inflammatory neutrophils and monocytes, followed by the accumulation of suppressive macrophages with the emergence of metastases. Spatial profiling revealed that metastasis-associated immune cells were present in the metastasis core, with the exception of TREM2+ regulatory macrophages uniquely enriched at the metastatic invasive margin, consistent across both murine models and human patient samples. These regulatory macrophages (Mreg) contribute to the formation of an immune-suppressive niche, cloaking tumor cells from immune surveillance. Our study provides a compendium of immune cell dynamics across metastatic stages and niches, informing the development of metastasis-targeting immunotherapies. SIGNIFICANCE: Temporal and spatial single-cell analysis of metastasis stages revealed new players in modulating immune surveillance and suppression. Our study highlights distinct populations of TREM2 macrophages as modulators of the microenvironment in metastasis, and as the key immune determinant defining metastatic niches, pointing to myeloid checkpoints to improve therapeutic strategies. This article is featured in Selected Articles from This Issue, p. 2489.
Assuntos
Neoplasias da Mama , Neoplasias Pulmonares , Camundongos , Humanos , Animais , Feminino , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias Pulmonares/patologia , Pulmão/patologia , Macrófagos , Microambiente Tumoral , Metástase Neoplásica/patologia , Glicoproteínas de Membrana , Receptores ImunológicosRESUMO
Mono-allelic germline disruptions of the transcription factor GATA2 result in a propensity for developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), affecting more than 85% of carriers. How a partial loss of GATA2 functionality enables leukemic transformation years later is unclear. This question has remained unsolved mainly due to the lack of informative models, as Gata2 heterozygote mice do not develop hematologic malignancies. Here we show that two different germline Gata2 mutations (TgErg/Gata2het and TgErg/Gata2L359V) accelerate AML in mice expressing the human hematopoietic stem cell regulator ERG. Analysis of Erg/Gata2het fetal liver and bone marrow-derived hematopoietic cells revealed a distinct pre-leukemic phenotype. This was characterized by enhanced transition from stem to progenitor state, increased proliferation, and a striking mitochondrial phenotype, consisting of highly expressed oxidative-phosphorylation-related gene sets, elevated oxygen consumption rates, and notably, markedly distorted mitochondrial morphology. Importantly, the same mitochondrial gene-expression signature was observed in human AML harboring GATA2 aberrations. Similar to the observations in mice, non-leukemic bone marrows from children with germline GATA2 mutation demonstrated marked mitochondrial abnormalities. Thus, we observed the tumor suppressive effects of GATA2 in two germline Gata2 genetic mouse models. As oncogenic mutations often accumulate with age, GATA2 deficiency-mediated priming of hematopoietic cells for oncogenic transformation may explain the earlier occurrence of MDS/AML in patients with GATA2 germline mutation. The mitochondrial phenotype is a potential therapeutic opportunity for the prevention of leukemic transformation in these patients.
Assuntos
Deficiência de GATA2 , Leucemia Mieloide Aguda , Síndromes Mielodisplásicas , Criança , Humanos , Camundongos , Animais , Deficiência de GATA2/genética , Síndromes Mielodisplásicas/patologia , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Medula Óssea/patologia , Células-Tronco Hematopoéticas/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Fator de Transcrição GATA2/genética , Fator de Transcrição GATA2/metabolismoRESUMO
BACKGROUND: The clinical presentation of coronavirus disease 19 (COVID-19) is the result of intricate interactions between the novel coronavirus and the immune system. In patients with hematologic malignancies (HM), these interactions dramatically change the clinical course and outcomes of COVID-19. SUMMARY: Patients with HM and COVID-19 are at an increased risk for prolonged viral shedding, more protracted and severe presentation, and death, even when compared to other immunocompromised hosts. HM (e.g., multiple myeloma, chronic lymphocytic leukemia) and anticancer treatments (e.g., anti-CD20 agents) that impair humoral immunity markedly increase the risk of severe COVID-19 as well as protracted viral shedding and possibly longer infectivity. Cytokine release syndrome (CRS) is an important player in the pathophysiology of severe and fatal COVID-19. Treatments targeting specific cytokines involved in CRS such as interleukin-6 and Janus kinase have proven beneficial in COVID-19 patients but were not assessed specifically in HM patients. Although neutropenia (as well as neutrophilia) was associated with increased COVID-19 mortality, granulocyte colony-stimulating factors were not beneficial in patients with COVID-19 and may have been associated with worse outcomes. Decreased levels of T lymphocytes and especially decreased CD4+ counts, and depletion of CD8+ lymphocytes, are a hallmark of severe COVID-19, and even more so among patients with HM, underlying the important role of T-helper dysfunction in severe COVID-19. In HM patients with intact cellular immunity, robust T-cell responses may compensate for an impaired humoral immune system. Further prospective studies are needed to evaluate the mechanisms of severe COVID-19 among patients with HM and assess the efficacy of new immunomodulating COVID-19 treatments in this population. KEY MESSAGES: Understanding the immunopathology of COVID-19 has greatly benefited from the previous research in patients with HM. So far, no COVID-19 treatments were properly evaluated in patients with HM. Patients with HM should be included in future RCTs assessing treatments for COVID-19.
Assuntos
COVID-19 , Neoplasias Hematológicas , Mieloma Múltiplo , Neutropenia , COVID-19/complicações , Neoplasias Hematológicas/complicações , Humanos , Imunidade , Mieloma Múltiplo/complicações , Neutropenia/complicaçõesRESUMO
Despite their key regulatory role and therapeutic potency, the molecular signatures of interactions between T cells and antigen-presenting myeloid cells within the tumor microenvironment remain poorly characterized. Here, we systematically characterize these interactions using RNA sequencing of physically interacting cells (PIC-seq) and find that CD4+PD-1+CXCL13+ T cells are a major interacting hub with antigen-presenting cells in the tumor microenvironment of human non-small cell lung carcinoma. We define this clonally expanded, tumor-specific and conserved T-cell subset as T-helper tumor (Tht) cells. Reconstitution of Tht cells in vitro and in an ovalbumin-specific αß TCR CD4+ T-cell mouse model, shows that the Tht program is primed in tumor-draining lymph nodes by dendritic cells presenting tumor antigens, and that their function is important for harnessing the antitumor response of anti-PD-1 treatment. Our molecular and functional findings support the modulation of Tht-dendritic cell interaction checkpoints as a major interventional strategy in immunotherapy.
Assuntos
Neoplasias Pulmonares , Microambiente Tumoral , Animais , Linhagem Celular Tumoral , Células Dendríticas , Inibidores de Checkpoint Imunológico/farmacologia , Neoplasias Pulmonares/terapia , Camundongos , Linfócitos T Auxiliares-IndutoresRESUMO
Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are prevalent liver conditions that underlie the development of life-threatening cirrhosis, liver failure and liver cancer. Chronic necro-inflammation is a critical factor in development of NASH, yet the cellular and molecular mechanisms of immune dysregulation in this disease are poorly understood. Here, using single-cell transcriptomic analysis, we comprehensively profiled the immune composition of the mouse liver during NASH. We identified a significant pathology-associated increase in hepatic conventional dendritic cells (cDCs) and further defined their source as NASH-induced boost in cycling of cDC progenitors in the bone marrow. Analysis of blood and liver from patients on the NAFLD/NASH spectrum showed that type 1 cDCs (cDC1) were more abundant and activated in disease. Sequencing of physically interacting cDC-T cell pairs from liver-draining lymph nodes revealed that cDCs in NASH promote inflammatory T cell reprogramming, previously associated with NASH worsening. Finally, depletion of cDC1 in XCR1DTA mice or using anti-XCL1-blocking antibody attenuated liver pathology in NASH mouse models. Overall, our study provides a comprehensive characterization of cDC biology in NASH and identifies XCR1+ cDC1 as an important driver of liver pathology.
Assuntos
Células Dendríticas/imunologia , Fígado Gorduroso/imunologia , Hepatopatia Gordurosa não Alcoólica/imunologia , Receptores de Quimiocinas/genética , Animais , Células da Medula Óssea/imunologia , Células da Medula Óssea/patologia , Reprogramação Celular/genética , Reprogramação Celular/imunologia , Células Dendríticas/patologia , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Feminino , Humanos , Fígado/imunologia , Fígado/patologia , Linfonodos/imunologia , Linfonodos/patologia , Masculino , Camundongos , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/patologia , Receptores de Quimiocinas/imunologia , Linfócitos T/imunologia , Linfócitos T/patologiaRESUMO
Tertiary lymphoid structures (TLS) are organized aggregates of B and T cells formed ectopically during different stages of life in response to inflammation, infection, or cancer. Here, we describe formation of structures reminiscent of TLS in the spinal cord meninges under several central nervous system (CNS) pathologies. After acute spinal cord injury, B and T lymphocytes locally aggregate within the meninges to form TLS-like structures, and continue to accumulate during the late phase of the response to the injury, with a negative impact on subsequent pathological conditions, such as experimental autoimmune encephalomyelitis. Using a chronic model of spinal cord pathology, the mSOD1 mouse model of amyotrophic lateral sclerosis, we further showed by single-cell RNA-sequencing that a meningeal lymphocyte niche forms, with a unique organization and activation state, including accumulation of pre-B cells in the spinal cord meninges. Such a response was not found in the CNS-draining cervical lymph nodes. The present findings suggest that a special immune response develops in the meninges during various neurological pathologies in the CNS, a possible reflection of its immune privileged nature.
Assuntos
Esclerose Lateral Amiotrófica/imunologia , Linfócitos B/imunologia , Imunidade , Meninges/imunologia , Traumatismos da Medula Espinal/imunologia , Linfócitos T/imunologia , Estruturas Linfoides Terciárias/imunologia , Doença Aguda , Animais , Doença Crônica , Modelos Animais de Doenças , Inflamação/imunologia , Linfonodos/imunologia , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pescoço , Vértebras Torácicas/lesõesRESUMO
Differentiation of CD4+ T cells into either follicular helper T (TFH) or type 1 helper T (TH1) cells influences the balance between humoral and cellular adaptive immunity, but the mechanisms whereby pathogens elicit distinct effector cells are incompletely understood. Here we analyzed the spatiotemporal dynamics of CD4+ T cells during infection with recombinant vesicular stomatitis virus (VSV), which induces early, potent neutralizing antibodies, or recombinant lymphocytic choriomeningitis virus (LCMV), which induces a vigorous cellular response but inefficient neutralizing antibodies, expressing the same T cell epitope. Early exposure of dendritic cells to type I interferon (IFN), which occurred during infection with VSV, induced production of the cytokine IL-6 and drove TFH cell polarization, whereas late exposure to type I IFN, which occurred during infection with LCMV, did not induce IL-6 and allowed differentiation into TH1 cells. Thus, tight spatiotemporal regulation of type I IFN shapes antiviral CD4+ T cell differentiation and might instruct vaccine design strategies.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Interferon Tipo I/metabolismo , Imunidade Adaptativa , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/classificação , Diferenciação Celular/imunologia , Feminino , Interleucina-6/biossíntese , Vírus da Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/patogenicidade , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise Espaço-Temporal , Linfócitos T Auxiliares-Indutores/imunologia , Células Th1/imunologia , Vírus da Estomatite Vesicular Indiana/imunologia , Vírus da Estomatite Vesicular Indiana/patogenicidade , Vírus da Estomatite Vesicular New Jersey/imunologia , Vírus da Estomatite Vesicular New Jersey/patogenicidadeRESUMO
Tumors are supported by cancer-associated fibroblasts (CAFs). CAFs are heterogeneous and carry out distinct cancer-associated functions. Understanding the full repertoire of CAFs and their dynamic changes as tumors evolve could improve the precision of cancer treatment. Here we comprehensively analyze CAFs using index and transcriptional single-cell sorting at several time points along breast tumor progression in mice, uncovering distinct subpopulations. Notably, the transcriptional programs of these subpopulations change over time and in metastases, transitioning from an immunoregulatory program to wound-healing and antigen-presentation programs, indicating that CAFs and their functions are dynamic. Two main CAF subpopulations are also found in human breast tumors, where their ratio is associated with disease outcome across subtypes and is particularly correlated with BRCA mutations in triple-negative breast cancer. These findings indicate that the repertoire of CAF changes over time in breast cancer progression, with direct clinical implications.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias de Mama Triplo Negativas , Animais , Fibroblastos Associados a Câncer/metabolismo , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Proteína A4 de Ligação a Cálcio da Família S100/genética , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
Dendritic cells (DC) are currently classified as conventional DCs (cDCs) and plasmacytoid DCs (pDCs). Through a combination of single-cell transcriptomic analysis, mass cytometry, in vivo fate mapping and in vitro clonal assays, here we show that, at the single-cell level, the priming of mouse hematopoietic progenitor cells toward the pDC lineage occurs at the common lymphoid progenitor stage, indicative of early divergence of the pDC and cDC lineages. We found the transcriptional signature of a pDC precursor stage, defined here, in the IL-7Rα+ common lymphoid progenitor population and identified Ly6D, IL-7Rα, CD81 and CD2 as key markers of pDC differentiation, which distinguish pDC precursors from cDC precursors. In conclusion, pDCs developed in the bone marrow from a Ly6DhiCD2hi lymphoid progenitor cell and differentiated independently of the myeloid cDC lineage.
Assuntos
Antígenos Ly/metabolismo , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Células Progenitoras Linfoides/citologia , Células Progenitoras Linfoides/metabolismo , Células Progenitoras Mieloides/citologia , Células Progenitoras Mieloides/metabolismo , Animais , Linfócitos B/imunologia , Linfócitos B/metabolismo , Biomarcadores , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Citometria de Fluxo , Proteínas Ligadas por GPI/metabolismo , Expressão Gênica , Perfilação da Expressão Gênica , Camundongos , TranscriptomaRESUMO
Lung development and function arises from the interactions between diverse cell types and lineages. Using single-cell RNA sequencing (RNA-seq), we characterize the cellular composition of the lung during development and identify vast dynamics in cell composition and their molecular characteristics. Analyzing 818 ligand-receptor interaction pairs within and between cell lineages, we identify broadly interacting cells, including AT2, innate lymphocytes (ILCs), and basophils. Using interleukin (IL)-33 receptor knockout mice and in vitro experiments, we show that basophils establish a lung-specific function imprinted by IL-33 and granulocyte-macrophage colony-stimulating factor (GM-CSF), characterized by unique signaling of cytokines and growth factors important for stromal, epithelial, and myeloid cell fates. Antibody-depletion strategies, diphtheria toxin-mediated selective depletion of basophils, and co-culture studies show that lung resident basophils are important regulators of alveolar macrophage development and function. Together, our study demonstrates how whole-tissue signaling interaction map on the single-cell level can broaden our understanding of cellular networks in health and disease.
Assuntos
Basófilos/metabolismo , Comunicação Celular , Impressão Genômica , Macrófagos Alveolares/metabolismo , Transcriptoma , Animais , Diferenciação Celular , Linhagem Celular Tumoral , Células Cultivadas , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Interleucina-33/metabolismo , Macrófagos Alveolares/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Análise de Célula ÚnicaRESUMO
T cell development and selection are coordinated in the thymus by a specialized niche of diverse stromal populations1-3. Although much progress has been made over the years in identifying the functions of the different cell types of the thymic stromal compartment, there is no comprehensive characterization of their diversity and heterogeneity. Here we combined massively parallel single-cell RNA-sequencing4,5, spatial mapping, chromatin profiling and gene targeting to characterize de novo the entire stromal compartment of the mouse thymus. We identified dozens of cell states, with thymic epithelial cells (TECs) showing the highest degree of heterogeneity. Our analysis highlights four major medullary TEC (mTEC I-IV) populations, with distinct molecular functions, epigenetic landscapes and lineage regulators. Specifically, mTEC IV constitutes a new and highly divergent TEC lineage with molecular characteristics of the gut chemosensory epithelial tuft cells. Mice deficient in Pou2f3, a master regulator of tuft cells, have complete and specific depletion of mTEC IV cells, which results in increased levels of thymus-resident type-2 innate lymphoid cells. Overall, our study provides a comprehensive characterization of the thymic stroma and identifies a new tuft-like TEC population, which is critical for shaping the immune niche in the thymus.
Assuntos
Células Epiteliais/citologia , Células Epiteliais/metabolismo , Interleucina-17/metabolismo , Interleucinas/metabolismo , Análise de Célula Única , Timo/citologia , Timo/imunologia , Animais , Epigênese Genética , Células Epiteliais/imunologia , Feminino , Humanos , Interleucina-17/biossíntese , Interleucinas/biossíntese , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Modelos Moleculares , Fatores de Transcrição/biossíntese , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína AIRERESUMO
Hematopoietic stem cells (HSCs) are considered a heterogeneous cell population. To further resolve the HSC compartment, we characterized a retinoic acid (RA) reporter mouse line. Sub-fractionation of the HSC compartment in RA-CFP reporter mice demonstrated that RA-CFP-dim HSCs were largely non-proliferative and displayed superior engraftment potential in comparison with RA-CFP-bright HSCs. Gene expression analysis demonstrated higher expression of RA-target genes in RA-CFP-dim HSCs, in contrast to the RA-CFP reporter expression, but both RA-CFP-dim and RA-CFP-bright HSCs responded efficiently to RA in vitro. Single-cell RNA sequencing (RNA-seq) of >1,200 HSCs showed that differences in cell cycle activity constituted the main driver of transcriptional heterogeneity in HSCs. Moreover, further analysis of the single-cell RNA-seq data revealed that stochastic low-level expression of distinct lineage-affiliated transcriptional programs is a common feature of HSCs. Collectively, this work demonstrates the utility of the RA-CFP reporter line as a tool for the isolation of superior HSCs.
Assuntos
Compartimento Celular , Ciclo Celular/genética , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Transcrição Gênica , Animais , Ciclo Celular/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Genoma , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Transplante de Células-Tronco Hematopoéticas , Células-Tronco Hematopoéticas/efeitos dos fármacos , Proteínas Luminescentes/metabolismo , Camundongos , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Transcriptoma/genética , Tretinoína/farmacologiaRESUMO
The dynamics of haematopoietic stem cell differentiation and the hierarchy of oligopotent stem cells in the bone marrow remain controversial. Here we dissect haematopoietic progenitor populations at single cell resolution, deriving an unbiased reference model of transcriptional states in normal and perturbed murine bone marrow. We define the signature of the naive haematopoietic stem cell and find a continuum of core progenitor states. Core cell populations mix transcription of pre-myeloid and pre-lymphoid programs, but do not mix erythroid or megakaryocyte programs with other fates. CRISP-seq perturbation analysis confirms our models and reveals that Cebpa regulates entry into all myeloid fates, while Irf8 and PU.1 deficiency block later differentiation towards monocyte or granulocyte fates. Our transcriptional map defines a reference network model for blood progenitors and their differentiation trajectories during normal and perturbed haematopoiesis.
Assuntos
Perfilação da Expressão Gênica/métodos , Hematopoese/genética , Células-Tronco Hematopoéticas/fisiologia , Análise de Célula Única/métodos , Transcriptoma , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Sistemas CRISPR-Cas , Linhagem da Célula/genética , Proliferação de Células/genética , Células Cultivadas , Senescência Celular/genética , Eritropoetina/farmacologia , Feminino , Filgrastim/farmacologia , Edição de Genes , Regulação da Expressão Gênica , Genótipo , Hematínicos/farmacologia , Hematopoese/efeitos dos fármacos , Células-Tronco Hematopoéticas/efeitos dos fármacos , Células-Tronco Hematopoéticas/metabolismo , Homeostase , Fatores Reguladores de Interferon/genética , Fatores Reguladores de Interferon/metabolismo , Camundongos Endogâmicos C57BL , Fenótipo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Recombinantes/farmacologia , Fatores de Tempo , Transativadores/genética , Transativadores/metabolismoRESUMO
Cellular functions are strongly dependent on surrounding cells and environmental factors. Current technologies are limited in their ability to characterize the spatial location and gene programs of cells in poorly structured and dynamic niches. We developed a method, NICHE-seq, that combines photoactivatable fluorescent reporters, two-photon microscopy, and single-cell RNA sequencing (scRNA-seq) to infer the cellular and molecular composition of niches. We applied NICHE-seq to examine the high-order assembly of immune cell networks. NICHE-seq is highly reproducible in spatial tissue reconstruction, enabling identification of rare niche-specific immune subpopulations and gene programs, including natural killer cells within infected B cell follicles and distinct myeloid states in the spleen and tumor. This study establishes NICHE-seq as a broadly applicable method for elucidating high-order spatial organization of cell types and their molecular pathways.
Assuntos
Linfócitos B/imunologia , Perfilação da Expressão Gênica/métodos , Células Matadoras Naturais/imunologia , Análise de Sequência de RNA/métodos , Análise de Célula Única/métodos , Animais , Genes Reporter/efeitos dos fármacos , Genômica/métodos , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Camundongos , Camundongos Transgênicos , Microscopia Confocal , Microscopia de Fluorescência por Excitação Multifotônica , Neoplasias/imunologia , Baço/imunologia , Baço/virologia , Viroses/imunologiaRESUMO
Monocytes are circulating, short-lived mononuclear phagocytes, which in mice and man comprise two main subpopulations. Murine Ly6C+ monocytes display developmental plasticity and are recruited to complement tissue-resident macrophages and dendritic cells on demand. Murine vascular Ly6C- monocytes patrol the endothelium, act as scavengers, and support vessel wall repair. Here we characterized population and single cell transcriptomes, as well as enhancer and promoter landscapes of the murine monocyte compartment. Single cell RNA-seq and transplantation experiments confirmed homeostatic default differentiation of Ly6C+ into Ly6C- monocytes. The main two subsets were homogeneous, but linked by a more heterogeneous differentiation intermediate. We show that monocyte differentiation occurred through de novo enhancer establishment and activation of pre-established (poised) enhancers. Generation of Ly6C- monocytes involved induction of the transcription factor C/EBPß and C/EBPß-deficient mice lacked Ly6C- monocytes. Mechanistically, C/EBPß bound the Nr4a1 promoter and controlled expression of this established monocyte survival factor.
Assuntos
Antígenos Ly/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Genômica , Monócitos/metabolismo , Animais , Biomarcadores , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Diferenciação Celular/genética , Análise por Conglomerados , Epigênese Genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Imunofenotipagem , Masculino , Camundongos , Camundongos Knockout , Células Precursoras de Monócitos e Macrófagos/classificação , Células Precursoras de Monócitos e Macrófagos/metabolismo , Monócitos/citologia , Monócitos/imunologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Fenótipo , Regiões Promotoras Genéticas , Ligação ProteicaRESUMO
Within the bone marrow, stem cells differentiate and give rise to diverse blood cell types and functions. Currently, hematopoietic progenitors are defined using surface markers combined with functional assays that are not directly linked with in vivo differentiation potential or gene regulatory mechanisms. Here, we comprehensively map myeloid progenitor subpopulations by transcriptional sorting of single cells from the bone marrow. We describe multiple progenitor subgroups, showing unexpected transcriptional priming toward seven differentiation fates but no progenitors with a mixed state. Transcriptional differentiation is correlated with combinations of known and previously undefined transcription factors, suggesting that the process is tightly regulated. Histone maps and knockout assays are consistent with early transcriptional priming, while traditional transplantation experiments suggest that in vivo priming may still allow for plasticity given strong perturbations. These data establish a reference model and general framework for studying hematopoiesis at single-cell resolution.